Insecticidal protein toxins from xenorhabdus

ABSTRACT

Proteins from the genus Xenorhabdus are toxic to insects upon oral exposure. These protein toxins can be applied to insect larvae food and plants for insect control.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This patent application claims priority from a U.S. ProvisionalPatent Application Serial No. 60/045,641 filed on May 5, 1997.

FIELD OF THE INVENTION

[0002] The present invention relates to toxins isolated from bacteriaand the use of said toxins as insecticides.

BACKGROUND OF THE INVENTION

[0003] In the past there has been interest in using biological agents asan option for pest management. One such method explored was thepotential of insect control using certain genera of nematodes.Nematodes, like those of the Steinernema and Heterorhabditis genera, canbe used as biological agents due in part to their transmissibleinsecticidal bacterial symbionts of the genera Xenorhabdus andPhotorhabdus, respectively. Upon entry into the insect, the nematodesrelease their bacterial symbionts into the insect hemolymph where thebacteria reproduce and eventually cause insect death. The nematode thendevelops and reproduces within the cadaver. Bacteria-containing nematodeprogeny exit the insect cadaver as infective juveniles which can theninvade additional larvae thus repeating the cycle leading to nematodepropagation. While this cycle is easily performed on a micro scale in alaboratory setting, adaptation to the macro level, as needed to beeffective as a general use insecticide, is difficult, expensive, andinefficient to produce, maintain, distribute and apply.

[0004] In addition to biological approaches to pest management such asnematodes, there are now pesticide control agents commercially availablethat are naturally derived. These naturally derived approaches can be aseffective as synthetic chemical approaches. One such naturally occurringagent is the crystal protein toxin produced by the bacteria Bacillusthuringiensis (Bt) These protein toxins have been formulated assprayable insect control agents. A more recent application of Bttechnology has been to isolate and transform into plants the genes thatproduce the toxins. Transgenic plants subsequently produce the Bt toxinsthereby providing insect control, (see U.S. Patent Nos. 5,380,831;5,567,600; and 5,567,862 to Mycogen in San Diego, Calif.).

[0005] Transgenic Bt plants are quite efficacious and usage is predictedto be high in some crops and areas. This has caused a concern thatresistance management issues may arise more quickly than withtraditional sprayable applications. Thus, it would be quite desirable todiscover other bacterial sources distinct from Bt which produce toxinsthat could be used in transgenic plant strategies, or could be combinedwith Bts to produce insect controlling transgenic plants.

[0006] It has been known in the art that bacteria of the genusXenorhabdus are symbiotically associated with the Steinernema nematode.Unfortunately, as reported in a number of articles, the bacteria onlyhad pesticidal activity when injected into insect larvae and did notexhibit biological activity when delivered orally (see Jarosz J. et al.“Involvement of Larvicidal Toxins in Pathogenesis of Insect Parasitismwith the Rhabditoid Nematodes, Steinernema Feltiae and HeterorhabditisBacteriophora” Entomophaga 36 (3) 1991 361-368; Balcerzak, Malgorzata“Comparative studies on parasitism caused by entomogenous nematodes,Steinernema feltiae and Heterorhabditis bacteriophors I. The roles ofthe nematode-bacterial complex, and of the associated bacteria alone, inpathogenesis” Acta Parasitologica Polonica, 1991, 36(4), 175-181).

[0007] For the reasons stated above it has been difficult to effectivelyexploit the insecticidal properties of the nematode or its bacterialsymbiont. Thus, it would be quite desirable to discover proteinaceousagents derived from Xenorhabdus bacteria that have oral activity so thatthe products produced therefrom could either be formulated as asprayable insecticide or the bacterial genes encoding said proteinaceousagents could be isolated and used in the production of transgenicplants. Until applicants' invention herein there was no knownXenorhabdus species or strains that produced protein toxin(s) havingoral activity.

SUMMARY OF THE INVENTION

[0008] The native toxins are protein complexes that are produced andsecreted by growing bacterial cells of the genus Xenorhabdus. Theprotein complexes, with a native molecular size ranging from about 800to 3000 kDa, can be separated by SDS-PAGE gel analysis into numerouscomponent proteins. The toxins exhibit significant toxicity uponexposure to a number of insects. Furthermore, toxin activity can bemodified by altering media conditions. In addition, the toxins havecharacteristics of being proteinaceous in that the activity thereof isheat labile and sensitive to proteolysis.

[0009] The present invention provides an easily administered functionalprotein.

[0010] The present invention also provides a method for deliveringinsecticidal toxins that are functionally active and effective againstmany orders of insects.

[0011] Objects, advantages, and features of the present invention willbecome apparent from the following specification.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012]FIG. 1 is a phenogram of Xenorhabdus strains as defined byrep-PCR. The upper axis of FIG. 1 measures the percentage similarity ofstrains based on scoring of rep-PCR products (i.e., 0.0 [no similarity]to 1.0 [100% similarity]). At the right axis, the numbers and lettersindicate the various strains tested. Vertical lines separatinghorizontal lines indicate the degree of relatedness (as read from theextrapolated intersection of the vertical line with the upper axis)between strains or groups of strains at the base of the horizontal lines(e.g., strain DEX1 is about 83% similar to strain X. nem).

DETAILED DESCRIPTION OF THE INVENTION

[0013] The present inventions are directed to discovery of a uniqueclass of functional protein toxins as defined herein produced bybacteria of the genus Xenorhabdus, said toxins having oral toxicityagainst insects. Xenorhabdus species/strains may be isolated from avariety of sources. One such source is entomopathogenic nematodes, moreparticularly nematodes of the genus Steinernema or from insect cadaversinfested by these nematodes. It is possible that other sources couldharbor Xenorhabdus bacteria that produce insecticidal toxins havingfunctional activity. Such sources in the environment could be eitherterrestrial or aquatic based.

[0014] The genus Xenorhabdus is taxonomically defined as a member of theFamily Enterobacteriaceae, although it has certain traits atypical ofthis family. For example, strains of this genus are typically nitratereduction negative, and catalase negative. Xenorhabdus has only recentlybeen subdivided to create a second genus; Photorhabdus which iscomprised of the single species Photorhabdus luminescens (previouslyXenorhabdus luminescens) (Boemare et al., 1993 Int. J. Syst. Bacteriol.43, 249-255). This differentiation is based on several distinguishingcharacteristics easily identifiable by the skilled artisan. Thesedifferences include the following: DNA-DNA characterization studies;phenotypic presence (Photorhabdus) or absence (Xenorhabdus) of catalaseactivity; presence (Photorhabdus) or absence (Xenorhabdus) ofbioluminescence; the Family of the nematode host in that Xenorhabdus isfound in Steinernematidae and Photorhabdus is found inHeterorhabditidae); as well as comparative, cellular fatty-acid analyses(Janse et al. 1990, Lett. Appl. Microbiol 10, 131-135; Suzuki et al.1990, J. Gen. Appl. Microbiol., 36, 393-401). In addition, recentmolecular studies focused on sequence (Rainey et al. 1995, Int. J. Syst.Bacteriol., 45, 379-381) and restriction analysis (Brunel et al., 1997,App. Environ. Micro., 63, 574-580) of 16S rRNA genes also support theseparation of these two genera. This change in nomenclature is reflectedin this specification, but in no way should a future change innomenclature alter the scope of the inventions described herein.

[0015] In order to establish that the strains disclosed herein werecomprised of Xenorhabdus strains, the strains were characterized basedon recognized traits which define Xenorhabdus species/strains anddifferentiate them from other Enterobacteriaceae and Photorhabdusspecies/strains. (Farmer, 1984 Bergey's Manual of Systemic BacteriologyVol. 1, pp. 510-511; Akhurst and Boemare 1988, J. Gen. Microbiol. 134,pp. 1835-1845; Boemare et al. 1993 int. J. Syst. Bacteriol. 43, pp.249-255, which are incorporated herein by reference). The expectedtraits for Xenorhabdus are the following: Gram stain negative rods,organism size of 0.3-2×2-10 μm, white to yellow/brown colonypigmentation, presence of inclusion bodies, absence of catalase,inability to reduce nitrate, absence of bioluminescence, ability touptake dye from medium, positive gelatin hydrolysis, growth onEnterobacteriaceae selective media, growth temperature below 37° C.,survival under anaerobic conditions, and motility.

[0016] Currently, the bacterial genus Xenorhabdus is comprised of fourrecognized species, Xenorhabdus nematophilus, Xenorhabdus poinarii,Xenorhabdus bovienii and Xenorhabdus beddingii (Brunel et al., 1997,App. Environ. Micro., 63, 574-580). A variety of related strains havebeen described in the literature (e.g., Akhurst and Boemare 1988 J. Gen.Microbiol., 134, 1835-1845; Boemare et al. 1993 Int. J. Syst. Bacteriol.43, pp. 249-255; Putz et al. 1990, Appl. Environ. Microbiol., 56,181-186, Brunel et al., 1997, App. Environ. Micro., 63, 574-580, Raineyet al. 1995, Int. J. Syst. Bacteriol., 45, 379-381). NumerousXenorhabdus strains have i| been characterized herein. Such strains andthe characteristics thereof are listed in Table 3 in the Examples. Thesestrains have been deposited with the Agricultural Research ServicePatent Culture Collection (NRRL) at 1815 North University Street Peoria,Ill. 61604 U.S.A. As can be seen in FIG. 1, these strains are diverse.It is not unforeseen that in the future there may be other Xenorhabdusspecies that will have some or all of the attributes of the describedspecies as well as some different characteristics that are presently notdefined as a trait(s) of Xenorhabdus. However, the scope of theinvention herein is to any Xenorhabdus species or strains which produceproteins as described herein that have functional activity as orallyactive insect control agents, regardless of other traits andcharacteristics. Further included within the inventions are the strainsspecified herein and any mutants or phase variants thereof.

[0017] There are several terms that are used herein that have aparticular meaning and are as follows:

[0018] By “functional activity” it is meant herein that the proteintoxins function as orally active insect control agents, that theproteins have a toxic effect, or are able to disrupt or deter insectfeeding which may or may not cause death of the insect. When an insectcomes into contact with an effective amount of toxin derived fromXenorhabdus delivered via transgenic plant expression, formulatedprotein compositions(s), sprayable protein composition(s), a bait matrixor other delivery system, the results are typically death of the insect,or the insects do not feed upon the source which makes the toxinsavailable to the insects.

[0019] By “native size” is meant the undenatured size of the proteintoxin or protein toxin subunit produced by the Xenorhabdus strain ofinterest prior to any treatment or modification. Native sizes ofproteins can be determined by a variety of methods available to theskilled artisan including but not limited to gel filtrationchromatography, agarose and polyacrylamide gel electrophoresis, massspectroscopy, sedimentation coefficients and the like. Treatment ormodifications to alter protein native size can be performed byproteolysis, mutagenesis, gene truncation, protein unfolding and othersuch techniques available to the artisan skilled in the art of proteinbiochemistry and molecular biology.

[0020] The protein toxins discussed herein are typically referred to as“insecticides”. By insecticides it is meant herein that the proteintoxins have a “functional activity” as further defined herein and areused as insect control agents.

[0021] The term “toxic” or “toxicity” as used herein is meant to conveythat the toxins produced by Xenorhabdus have “functional activity” asdefined herein.

[0022] The term “Xenorhabdus toxin” is meant to include any proteinproduced by a Xenorhabdus microorganism strain having functionalactivity against insects, where the Xenorhabdus toxin could beformulated as a sprayable composition, expressed by a transgenic plant,formulated as a bait matrix, delivered via a baculovirus, a plant RNAviral based system, or delivered by any other applicable host ordelivery system. It is also meant to include any sequence of aminoacids, polypeptides peptide fragment or other protein preparation,whether derived in whole or in part from natural or synthetic sourceswhich demonstrates the ability to exhibit functional activity asdisclosed herein. Typically, a Xenorhabdus toxin will be derived inwhole or in part from a Xenorhabdus bacterial source.

[0023] The term “Xenorhabdus toxin” is also meant to include modifiedamino acid sequences, such as sequences which have been mutated,truncated, increased and the like, as well as such sequences which arepartially or wholly artificially synthesized. Xenorhabdus toxins andnucleic acid sequences encoding said toxins may be obtained by partialor homogenous purification of bacterial extracts, N-terminal or internalamino acid sequence information, protein modeling, nucleic acid probes,antibody preparations, or sequence comparison. Once a purified orpartially purified Xenorhabdus toxin is obtained, it may be used toobtain other Xenorhabdus toxins by immunoprecipitation involving theformation of an antigen:antibody immunocomplex thereby allowing recoveryof the new toxin which reacts thereto. Once the nucleic acid sequenceencoding a Xenorhabdus toxin is obtained, it may be employed in probesfor further screening or used in genetic engineering constructs fortranscription or transcription and translation in host cells.

[0024] Fermentation broths from selected strains reported in Table 3were used to examine the following: breadth of insecticidal toxinshaving functional activity produced by the Xenorhabdus genus, thefunctional spectrum of these toxins, and the protein components of saidtoxins. The strains characterized herein have been shown to have oraltoxicity against a variety of insect orders. Such insect orders includebut are not limited to Coleoptera, Lepidoptera, Diptera, and Acarina.

[0025] As with other bacterial toxins, the mutation rate of bacteria ina population may result in the variation of the sequence of toxin genes.Toxins of interest here are those which produce proteins havingfunctional activity against a variety of insects upon exposure, asdescribed herein. Preferably, the toxins are active against Lepidoptera,Coleoptera, Diptera, and Acarina. The inventions herein are intended tocapture the protein toxins homologous to protein toxins produced by thestrains herein and any derivative strains thereof, as well as any otherprotein toxins produced by Xenorhabdus that have functional activity.These homologous proteins may differ in sequence, but do not differ infunctional activity from those toxins described herein. Homologoustoxins are meant to include protein complexes of between 100 kDa to 3500kDa and are comprised of at least one subunit, where a subunit is apeptide which may or may not be the same as the other subunit.

[0026] The toxins described herein are quite unique in that the toxinshave functional activity, which is key to developing an insectmanagement strategy. In developing an insect management strategy, it ispossible to delay or circumvent the protein degradation process byinjecting a protein directly into an organism, avoiding its digestivetract. In such cases, the protein administered to the organism willretain its function until it is denatured, non-specifically degraded, oreliminated by the immune system in higher organisms. Injection intoinsects of an functional toxin has potential application only in thelaboratory.

[0027] The discovery that the functional protein toxins herein exhibittheir activity after oral ingestion or contact with the toxins permitsthe development of an insect management plan based solely on the abilityto incorporate the protein toxins into the insect diet. Such a plancould result in the production of insect baits.

[0028] The Xenorhabdus toxins may be administered to insects in both apurified and non-purified form. The toxins may also be delivered inamounts from about 1 to about 1000 mg/liter of broth. This may vary uponformulation condition, conditions of the inoculum source, techniques forisolation of the toxin, and the like. The toxins found herein can beadministered as a sprayable insecticide. Fermentation broth fromXenorhabdus can be produce, diluted, or if needed, be concentrated about100 to 1000-fold using ultrafiltration or other techniques available tothe skilled artisan. Treatments can be applied with a syringe sprayer, atrack sprayer or any such equipment available to the skilled artisanwherein the broth is applied to the plants. After treatments, broths canbe tested by applying the insect of choice to said sprayed plant and canthe be scored for damage to the leaves. If necessary, adjuvants andphoto-protectants can be added to increase toxin-environmentalhalf-life. In a laboratory setting, broth, dilutions, or concentratesthereof can be applied using methods available to the skilled artisan.Afterwards, the material can be allowed to dry and insects to be testedare applied directly to the appropriate plant tissue. After one week,plants can be scored for damage using a modified Guthrie Scale (Koziel,M. G., Beland, G. L., Bowman, C., Carozzi, N. B., Crenshaw, R.,Crossland, L., Dawson, J., Desai, N., Hill, M., Kadwell, S., Launis, K.,Lewis, K., Maddox, D., McPherson, K., Meghji, M. Z., Merlin, E., Rhodes,R., Warren, G. W., Wright, M. and Evola, S. V. 1993). In this manner,broth or other protein containing fractions may confer protectionagainst specific insect pests when delivered in a sprayable formulationor when the gene or derivative thereof, encoding the protein or partthereof, is delivered via a transgenic plant or microbe.

[0029] The toxins may be administered as a secretion or cellular proteinoriginally expressed in a heterologous prokaryotic or eukaryotic host.Bacteria are typically the hosts in which proteins are expressed.Eukaryotic hosts could include but are not limited to plants, insectsand yeast. Alternatively, the toxins may be produced in bacteria ortransgenic plants in the field or in the insect by a baculovirus vector.Typically, insects will be exposed to toxins by incorporating one ormore of said toxins into the food/diet of the insect.

[0030] Complete lethality to feeding insects is preferred, but is notrequired to achieve functional activity. If an insect avoids the toxinor ceases feeding, that avoidance will be useful in some applications,even if the effects are sublethal or lethality is delayed or indirect.For example, if insect resistant transgenic plants are desired, thereluctance of insects to feed on the plants is as useful as lethaltoxicity to the insects since the ultimate objective is protection ofinsect-induced plant damage rather than insect death.

[0031] There are many other ways in which toxins can be incorporatedinto an insect's diet. For example, it is possible to adulterate thelarval food source with the toxic protein by spraying the food with aprotein solution, as disclosed herein. Alternatively, the purifiedprotein could be genetically engineered into an otherwise harmlessbacterium, which could then be grown in culture, and either applied tothe food source or allowed to reside in the soil in an area in whichinsect eradication was desirable. Also, the protein could be geneticallyengineered directly into an insect food source. For instance, the majorfood source for many insect larvae is plant material. Therefore thegenes encoding Xenorhabdus toxins can be transferred to plant materialso that said plant material expresses the toxin of interest.

[0032] Transfer of the functional activity to plant or bacterial systemsrequires nucleic acid sequences encoding the amino acid sequences forthe Xenorhabdus toxins integrated into a protein expression vectorappropriate to the host in which the vector will reside. One way toobtain a nucleic acid sequence encoding a protein with functionalactivity is to isolate the native genetic material from the bacterialspecies or Xenorhabdus species which produce the toxins, usinginformation deduced from the toxin's amino acid sequence, large portionsof which are disclosed herein.

[0033] There are also many different fermentation conditions that canaffect the amount or types of toxins produced by Xenorhabdus. Severaldifferent factors can be varied by the skilled artisan to optimize toxinproduction for increased or altered toxin activity. Such factors includebut are not limited to aeration of media, temperature, mediaconstituents such as phosphate, carbon sources, minerals, vitamins,sugars, nitrogen sources, pH and the like. Additional factors alsoinclude harvest time and the phase variant of the bacteria used.

[0034] Once broth containing toxin has been produce, there are manypurification technique and chromatographic media available to the personskilled in the art of protein biochemistry to allow purification ofXenorhabdus toxins. After each and every step, fractions can be assayedto find those particular fractions having the functional activity ofinterest as described herein. For example, protein toxins can beenriched in the broth by centrifugation, membrane separation, and thelike to form a highly enriched, concentrated solution of toxin beingpredominantly comprised of proteins having a native size greater than orequal to 100 kDa. The proteins can then fractionated by ion exchangechromatography where upon they are separated based on overall ioniccharge. Again, fractions obtained therefrom can be assayed against avariety of insects as described herein to find those fractions havingthe protein toxins of interest. Said proteins can then be separatedbased on native size using gel filtration-size exclusion chromatographyand the like. Typically, said fractions having functional activityappear to elute from gel filtration columns in a manner suggesting thatthe native toxin complex is about 500 kDa to about 3,250 kDa, preferablyabout 750 kDa to about 3000 kDa, with those in the range of about 800kDa to about 1100 kDa being most preferred. Fractions containing thetoxins of interest can then be further purified by using quantitativeion exchange, quantitative gel filtration, hydrophobic chromatography,isoelectric focusing and the like to again isolate highly enriched andpurified toxin fractions. The manner and order of protein purificationas described herein is exemplary only, thus other techniques andapproaches used by the skilled artisan to enrich and isolate Xenorhabdustoxins are within the scope of this invention.

[0035] When applied to SDS-PAGE analysis, fractions containing highlevels of Xenorhabdus toxin activity are shown to contain variousprotein subunits as taught in the Examples herein. Typically, theprotein subunits are between about 20 kDa to about 350 kDa; betweenabout 130 kDa to about 300 kDa; between about 200 kDa to about 220 kDa;about 40 kDa to about 80 kDa; and about 20 kDa to about 40 kDa.

[0036] Given the few bands provided in the SDS-PAGE, immediate effortsto obtain the corresponding amino acid and/or nucleic acid sequencesthereto are possible in accordance with methods familiar to thoseskilled in the art. From such sequences, Xenorhabdus toxins may befurther confirmed with expression in controlled systems, such as E. coliand the like. In addition, said sequences allow the production ofantibodies recognizing said toxins which can then be used to : identifyrelated Xenorhabdus toxin in other bacterial systems using methodsavailable to the skilled artisan.

[0037] Amino acid sequences of fragments corresponding to partially orfully purified protein preparations may be obtainable through digestionwith a protease, such as trypsin, and sequencing of resulting peptidefragments. Amino acid are disclosed herein. Said sequences can be usedto design oligonucleotides using the genetic code through reversetranslation. DNA sequences can then be chosen for use in PolymeraseChain Reactions (PCR) using genomic DNA isolated from Xenorhabdusbacterial cells. The resulting PCR-generated sequences can then be usedas labeled probes in screening genomic libraries. In this manner, thefull length clones corresponding to the Xenorhabdus toxin proteins seenon the SDS-PAGE may be recovered if desired. Other Xenorhabdus toxingenes may be obtained by screening genomic libraries from otherXenorhabdus species and other bacteria in the family Enterobacteriaceae.

[0038] The complete genomic sequence of a Xenorhabdus toxin may beobtained by the screening of a genomic or cosmid library with a probe.Probes can be considerably shorter than the entire gene sequence, butshould be at least about 10, preferably at least 15, more preferably atleast 20 or so nucleotides in length. Longer oligonucleotides are alsouseful, up to the full length of the gene encoding the polypeptide ofinterest. Both DNA and RNA can be used as probes. In use, probes aretypically labeled with ³²P, biotinylated, and the like in a manner thatallows for detection thereof. Said probes are often incubated withsingle stranded DNA from the source of which the gene is desired.Hybridization, or the act of the probe binding to the DNA, is detectedusually after hybridization using nitrocellulose paper or nylonmembranes by means of the label on said probe. Hybridization techniquesare well known to the person skilled in the art of molecular biology.Thus Xenorhabdus toxin genes may be isolated.

[0039] Other Xenorhabdus toxin genes or nucleic acid sequences areobtainable from amino acid sequences provided herein. “Obtainable”refers to those Xenorhabdus toxins and genes thereof which havesufficiently similar sequences or “homology” to that of the nativesequences of this invention to provide a orally active functional toxin.One skilled in the art will readily recognize that antibodypreparations, nucleic acid probes (DNA and RNA) and the like may beprepared using the amino acid sequences disclosed herein and used toscreen and recover other Xenorhabdus toxin nucleic acid sequences fromother sources. Thus, sequences homologously related to or derivations ofXenorhabdus toxins disclosed herein are considered obtainable from thepresent invention.

[0040] “Homologously related” includes those nucleic acid and amino acidsequences which are identical or conservatively substituted as comparedto the native sequence. Typically, a homologously related nucleic acidsequence will show at least about 60% homology, and more preferably atleast about 70% homology to the probes created from using the amino acidsequences disclosed herein and those nucleic acid sequences obtainedtherefrom using those methods and techniques as disclosed herein.Homology is determined upon comparison of sequence information, e.g.,nucleic acid or amino acid or through hybridization reactions. Homologyis also intended to include conservative amino acid substitutions, whichare will known in the art. Conservative amino acid substitutions includeglutamic acid/aspartic acid; valine/isoleucine/leucine;serine/threonine; arginine/lysine; glutamine/asparagine; or any suchsubstitution that results in no significant change in functionalactivity of said toxin when compared to the native toxin. Significantchange as used herein is defined as at least a 50% change in activitybased on molar amounts compared to said native toxin.

[0041] It is within the scope of the invention as disclosed herein thattoxins may be truncated and still retain functional activity. By“truncated toxin” is meant that a portion of a toxin protein may becleaved and yet still exhibit activity after cleavage. Cleavage can beachieved by proteases inside or outside of the insect gut. Furthermore,effectively cleaved proteins can be produced using molecular biologytechniques wherein the DNA bases encoding said toxin are removed eitherthrough digestion with restriction endonucleases or other techniquesavailable to the skilled artisan. After truncation, said proteins can beexpressed in heterologous systems such as E. coli, baculoviruses,plant-based viral systems, yeast and the like and then placed in insectassays as disclosed herein to determine activity. Truncated toxins havebeen successfully produced with several insecticidal protein toxins inthat several proteins have been shown in the art to retain functionalactivity while having less than the entire, full length protein present.Said truncated proteins having insecticidal activity include insectjuvenile hormone esterase (U.S. Pat. No. 5,674,485 to the Regents of theUniversity of California; and the insecticidal toxin isolated from thebacterium Bacillus thuringiensis (Adang et al., Gene 36:289-300 (1985)“Characterized full-length and truncated plasmid clones of the crystalprotein of Bacillus thuringiensis subsp kurstaki HD-73 and theirtoxicity to Manduca sexta)”. As used herein, the term “Xenorhabdustoxin” is also meant to include truncated versions thereof havingfunctional activity.

[0042] Recombinant constructs containing a nucleic acid sequenceencoding a Xenorhabdus toxin and heterologous nucleic acid sequences ofinterest may be prepared. By heterologous is meant any sequence which isnot naturally found joined to the synthase sequence. Hence, bydefinition, a sequence joined to sequence not naturally found in aXenorhabdus toxin is considered to be heterologous.

[0043] Constructs may be designed to produce Xenorhabdus toxins ineither prokaryotic or eukaryotic cells. The expression of a Xenorhabdustoxin in a plant cell is of special interest. Moreover, the nucleic acidsequence encoding a Xenorhabdus toxin may be integrated into a planthost genome. By transcribing and translating a nucleic acid sequenceencoding a Xenorhabdus toxin in a plant host, said plant is expected toexhibit properties whereby insects are discouraged from feeding. Asstated herein, it is not necessary for an functional agent to exhibitinsect mortality to be effective at controlling insects.

[0044] To obtain high expression of heterologous genes in plants it maybe preferred to reengineer said genes so that they are more efficientlyexpressed in the cytoplasm of plant cells. Maize is one such plant whereit may be preferred to reengineer the heterologous gene(s) prior totransformation to increase the expression level thereof in said plant.Therefore, an additional step in the design of genes encoding aXenorhabdus toxin is the designed reengineering of a heterologous genefor optimal expression.

[0045] One reason for the reengineering a Xenorhabdus toxin forexpression in maize is due to the non-optimal G+C content of the nativegene. For example, the very low G+C content of many native bacterialgene(s) (and consequent skewing towards high A+T content) results in thegeneration of sequences mimicking or duplicating plant gene controlsequences that are known to be highly A+T rich. The presence of someA+T-rich sequences within the DNA of gene(s) introduced into plants(e.g., TATA box regions normally found in gene promoters) may result inaberrant transcription of the gene(s). On the other hand, the presenceof other regulatory sequences residing in the transcribed mRNA (e.g.,polyadenylation signal sequences (AAUAAA), or sequences complementary tosmall nuclear RNAs involved in pre-mRNA splicing) may lead to RNAinstability. Therefore, one goal in the design of genes encoding aXenorhabdus toxin for maize expression, more preferably referred to asplant optimized gene(s), is to generate a DNA sequence having a higherG+C content, and preferably one close to that of maize genes coding formetabolic enzymes. Another goal in the design of the plant optimizedgene(s) encoding a Xenorhabdus toxin is to generate a DNA sequence inwhich the sequence modifications do not hinder translation.

[0046] The table below (Table 1) illustrates how high the G+C content isin maize. For the data in Table 1, coding regions of the genes wereextracted from GenBank (Release 71) entries, and base compositions werecalculated using the MacVector™ program (IBI, New Haven, Conn.). Intronsequences were ignored in the calculations.

[0047] Due to the plasticity afforded by the redundancy of the geneticcode (i.e., some amino acids are specified by more than one codon),evolution of the genomes in different organisms or classes of organismshas resulted in differential usage of redundant codons. This “codonbias” is reflected in the mean base composition of protein codingregions. For example, organisms with relatively low G+C contents utilizecodons having A or T in the third position of redundant codons, G or Cin the third position. It is thought that the presence of “minor” codonswithin a mRNA may reduce the absolute translation rate of that mRNA,especially when the relative abundance of the charged tRNA correspondingto the minor codon is low. An extension of this is that the diminutionof translation rate by individual minor codons would be at leastadditive for multiple minor codons. Therefore, mRNAs having highrelative contents of minor codons would have correspondingly lowtranslation rates. This rate would be reflected by subsequent low levelsof the encoded protein.

[0048] In reengineering genes encoding a Xenorhabdus toxin for maizeexpression, the codon bias of the plant has been determined. The codonbias for maize is the statistical codon distribution that the plant usesfor coding its proteins and the preferred codon usage is shown in Table2. After determining the bias, the percent frequency of the codons inthe gene(s) of interest is determined. The primary codons preferred bythe plant should be determined as well as the second and third choice ofpreferred codons. Afterwards, the amino acid sequence of the Xenorhabdustoxin of interest is reverse translated so that the resulting nucleicacid sequence codes for exactly the same protein as the native genewanting TABLE 1 Compilation of G + C contents of protein coding regionsof maize genes. Protein Class^(a) Range % G + C Mean % G + C^(b)Metabolic Enzymes (76) 44.4-75.3 59.0 (±8.0) Structural Proteins (18)48.6-70.5 63.6 (±6.7) Regulatory Proteins (5) 57.2-68.9 62.0 (±4.9)Uncharacterized Proteins (9) 41.5-70.3 64.3 (±7.2) All Proteins (108)44.4-75.3 60.8 (±5.2)

[0049] to be heterologously expressed. The new DNA sequence is designedusing codon bias information so that it corresponds to the mostpreferred codons of the desired plant. The new sequence is then analyzedfor restriction enzyme sites that might have been created by themodification. The identified sites are further modified by replacing thecodons with second or third choice with preferred codons. Other sites inthe sequence which could affect transcription or translation of the geneof interest are the exon:intron 5′ or 3′ junctions, poly A additionsignals, or RNA polymerase termination signals. The sequence is furtheranalyzed and modified to reduce the frequency of TA or GC doublets. Inaddition to the doublets, G or C sequence blocks that have more thanabout four residues that are the same can affect transcription of thesequence. Therefore, these blocks are also modified by replacing thecodons of first or second choice, etc. with the next preferred codon ofchoice.

[0050] It is preferred that the plant optimized gene(s) encoding aXenorhabdus toxin contain about 63% of first choice codons, betweenabout 22% to about 37% second choice codons, and between about 15% toabout 0% third choice codons, wherein the total percentage is 100%. Mostpreferred the plant optimized gene(s) contains about 63% of first choicecodons, at least about 22% second choice codons, about 7.5% third choicecodons, and about 7.5% fourth choice codons, wherein the totalpercentage is 100%. The preferred codon usage for engineering genes formaize expression are shown in Table 2. The method described aboveenables one skilled in the art to modify gene(s) that are foreign to aparticular plant so that the genes are optimally expressed in plants.The method is further illustrated in pending PCT application WO97/13402, which is incorporated herein by reference.

[0051] In order to design plant optimized genes encoding a Xenorhabdustoxin, the amino acid sequence of said protein is reverse translatedinto a DNA sequence utilizing a non-redundant genetic code establishedfrom a codon bias table compiled for the gene sequences for theparticular plant, as shown in Table 2. The resulting DNA sequence, whichis completely homogeneous in codon usage, is further modified toestablish a DNA sequence that, besides having a higher degree of codondiversity, also contains strategically placed restriction enzymerecognition sites, desirable base composition, and a lack of sequencesthat might interfere with transcription of the gene, or translation ofthe product mRNA.

[0052] In another aspect of the invention, genes encoding theXenorhabdus toxin are expressed from transcriptional units inserted intothe plant genome. Preferably, said transcriptional units are recombinantvectors capable of stable integration into the plant genome andselection of transformed plant lines expressing mRNA encoding for saiddesaturase proteins are expressed either by constitutive or induciblepromoters in the plant cell. Once expressed, the mRNA is translated intoproteins, thereby incorporating amino acids of interest into protein.The genes encoding a Xenorhabdus toxin expressed in the plant cells canbe under the control of a constitutive promoter, a tissue-specificpromoter or an inducible promoter as described herein.

[0053] Several techniques exist for introducing foreign recombinantvectors into plant cells, and for obtaining plants that stably maintainand express the introduced gene. Such techniques include acceleration ofgenetic material coated onto microparticles directly into cells (U.S.Pat. No. 4,945,050 to Cornell and U.S. Pat. No. 5,141,131 to DowElanco,now Dow AgroSciences, LLC) In addition, plants may be transformed usingAgrobacterium TABLE 2 Preferred amino acid codons for proteins expressedin maize. Amino Acid Codon* Alanine GCC/GCG Cysteine TGC/TGT AsparticAcid GAC/GAT Glutamic Acid GAG/GAA Phenylalanine TTC/TTT Glycine GGC/GGGHistidine CAC/CAT Isoleucine ATC/ATT Lysine AAG/AAA Leucine CTG/CTCMethionine ATG Asparagine AAC/AAT Proline CCG/CCA Glutamine CAG/CAAArginine AGG/CGC Serine AGC/TCC Threonine ACC/ACG Valine GTG/GTCTryptophan TGG Tyrosine TAC/TAT Stop TGA/TAG

[0054] technology, see U.S. Pat. No. 5,177,010 to University of Toledo,U.S. Pat. No. 5 5,104,310 to Texas A&M, European Patent Application0131624B1, European Patent Applications 120516, 159418B1 and 176,112 toSchilperoot, U.S. Pat. Nos. 5,149,645, 5,469,976, 5,464,763 and4,940,838 and 4,693,976 to Schilperoot, European Patent Applications116718, 290799, 320500 all to Max Planck, European Patent Applications604662,627752 and U.S. Pat. No.5,591,616 to Japan Tobacco, EuropeanPatent Applications 0267159, and 0292435 and U.S. Pat. No. 5,231,019 allto Ciba Geigy, now Novartis, U.S. Pat. Nos. 5,463,174 and 4,762,785 bothto Calgene, and U.S. Pat. Nos. 5,004,863 and 5,159,135 both toAgracetus. Other transformation technology includes whiskers technology,see U.S. Pat. Nos. 5,302,523 and 5,464,765 both to Zeneca.Electroporation technology has also been used to transform plants, seeWO 87/06614 to Boyce Thompson Institute, U.S. Pat Nos. 5,472,869 and5,384,253 both to Dekalb, WO9209696 and WO9321335 both to Plant GeneticSystems. Furthermore, viral vectors can also be used in producetransgenic plants expressing the protein of interest. For example,monocotyledonous plant can be transformed with a viral vector using themethods described in U.S. Pat No. 5,569,597 to Mycogen and Ciba-Giegy,now Novartis, as well as U.S. Pat Nos. 5,589,367 and 5,316,931, both toBiosource. All of these transformation patents and publications areincorporated herein by reference.

[0055] As mentioned previously, the manner in which the DNA construct isintroduced into the plant host is not critical to this invention. Anymethod which provides for efficient transformation may be employed. Forexample, various methods for plant cell transformation are describedherein and include the use of Ti or Ri-plasmids and the like to performAgrobacterium mediated transformation. In many instances, it will bedesirable to have the construct used for transformation bordered on oneor both sides by T-DNA borders, more specifically the right border. Thisis particularly useful when the construct uses Agrobacterium tumefaciensor Agrobacterium rhizogenes as a mode for transformation, although T-DNAborders may find use with other modes of transformation. WhereAgrobacterium is used for plant cell transformation, a vector may beused which may be introduced into the host for homologous recombinationwith T-DNA or the Ti or Ri plasmid present in the host. Introduction ofthe vector may be performed via electroporation, tri-parental mating andother techniques for transforming gram-negative bacteria which are knownto those skilled in the art. The manner of vector transformation intothe Agrobacterium host is not critical to w this invention. The Ti or Riplasmid containing the T-DNA for recombination may be capable orincapable of causing gall formation, and is not critical to saidinvention so long as the vir genes are present in said host.

[0056] In some cases where Agrobacterium is used for transformation, theexpression construct being within the T-DNA borders will be insertedinto a broad spectrum vector such as pRK2 or derivatives thereof asdescribed in Ditta et al., (PNAS USA (1980) 77:7347-7351 and EPO 0 120515, which are incorporated herein by reference. Included within theexpression construct and the T-DNA will be one or more markers asdescribed herein which allow for selection of transformed Agrobacteriumand transformed plant cells. The particular marker employed is notessential to this invention, with the preferred marker depending on thehost and construction used.

[0057] For transformation of plant cells using Agrobacterium, explantsmay be combined and incubated with the transformed Agrobacterium forsufficient time to allow transformation thereof. After transformation,the agrobacteria are killed by selection with the appropriate antibioticand plant cells are cultured with the appropriate selective medium. Oncecalli are formed, shoot formation can be encourage by employing theappropriate plant hormones according to methods well known in the art ofplant tissue culturing and plant regeneration. However, a callusintermediate stage is not always necessary. After shoot formation, saidplant cells can be transferred to medium which encourages root formationthereby completing plant regeneration. The plants may then be grown toseed and said seed can be used to establish future generations.Regardless of transformation technique, the gene encoding a Xenorhabdustoxin is preferably incorporated into a gene transfer vector adapted toexpress said gene in a plant cell by including in the vector a plantpromoter regulatory element, as well as 3′ non-translatedtranscriptional termination regions such as Nos and the like.

[0058] In addition to numerous technologies for transforming plants, thetype of tissue which is contacted with the foreign genes may vary aswell. Such tissue would include but would not be limited to embryogenictissue, callus tissue types I, II, and III, hypocotyl, meristem, roottissue and the like. Almost all plant tissues may be transformed duringdedifferentiation using appropriate techniques described herein.

[0059] Another variable is the choice of a selectable marker. Preferencefor a particular marker is at the discretion of the artisan, but any ofthe following selectable markers may be used along with any other genenot listed herein which could function as a selectable marker. Suchselectable markers include but are not limited to aminoglycosidephosphotransferase gene of transposon Tn5 (Aph II) which encodesresistance to the antibiotics kanamycin, neomycin and G418, as well asthose genes which encode for resistance or tolerance to glyphosate;hygromycin; methotrexate; phosphinothricin (bialophos); imidazolinones,sulfonylureas and triazolopyrimidine herbicides, such as chlorsulfuron;bromoxynil, dalapon and the like.

[0060] In addition to a selectable marker, it may be desirous to use areporter gene. In some instances a reporter gene may be used with orwithout a selectable marker. Reporter genes are genes which aretypically not present in the recipient organism or tissue and typicallyencode for proteins resulting in some phenotypic change or enzymaticproperty. Examples of such genes are provided in K. Wising et al. Ann.Rev. Genetics, 22, 421 (1988), which is incorporated herein byreference. Preferred reporter genes include the beta-glucuronidase (GUS)of the uidA locus of E. coli, the chloramphenicol acetyl transferasegene from Tn9 of E. coli, the green fluorescent protein from thebioluminescent jellyfish Aequorea victoria, and the luciferase genesfrom firefly Photinus pyralis. An assay for detecting reporter geneexpression may then be performed at a suitable time after said gene hasbeen introduced into recipient cells. A preferred such assay entails theuse of the gene encoding beta-glucuronidase (GUS) of the uidA locus ofE. coli as described by Jefferson et al., (1987 Biochem. Soc. Trans. 15,17-19) to identify transformed cells.

[0061] In addition to plant promoter regulatory elements, promoterregulatory elements from a variety of sources can be used efficiently inplant cells to express foreign genes. For example, promoter regulatoryelements of bacterial origin, such as the octopine synthase promoter,the nopaline synthase promoter, the mannopine synthase promoter;promoters of viral origin, such as the cauliflower mosaic virus (35S and19S), 35T (which is a re-engineered 35S promoter, see PCT/US96/1682; WO97/13402 published Apr. 17, 1997) and the like may be used. Plantpromoter regulatory elements include but are not limited toribulose-1,6-bisphosphate (RUBP) carboxylase small subunit (ssu),beta-conglycinin promoter, beta-phaseolin promoter, ADH promoter,heat-shock promoters and tissue specific promoters. Other elements suchas matrix attachment regions, scaffold attachment regions, introns,enhancers, polyadenylation sequences and the like may be present andthus may improve the transcription efficiency or DNA integration. Suchelements may or may not be necessary for DNA function, although they canprovide better expression or functioning of the DNA by affectingtranscription, mRNA stability, and the like. Such elements may beincluded in the DNA as desired to obtain optimal performance of thetransformed DNA in the plant. Typical elements include but are notlimited to Adh-intron 1, Adh-intron 6, the alfalfa mosaic virus coatprotein leader sequence, the maize streak virus coat protein leadersequence, as well as others available to a skilled artisan. Constitutivepromoter regulatory elements may also be used thereby directingcontinuous gene expression in all cells types and at all times (e.g.,actin, ubiquitin, CaMV 35S, and the like). Tissue specific promoterregulatory elements are responsible for gene expression in specific cellor tissue types, such as the leaves or seeds (e.g., zein, oleosin,napin, ACP, globulin and the like) and these may also be used.

[0062] Promoter regulatory elements may also be active during a certainstage of the plants' development as well as active in plant tissues andorgans. Examples of such include but are not limited to pollen-specific,embryo specific, corn silk specific, cotton fiber specific, rootspecific, seed endosperm specific promoter regulatory elements and thelike. Under certain circumstances it may be desirable to use aninducible promoter regulatory element, which is responsible forexpression of genes in response to a specific signal, such as: physicalstimulus (heat shock genes); light (RUBP carboxylase); hormone (Em);metabolites; chemical; and stress. Other desirable transcription andtranslation elements that function in plants may be used. Numerousplant-specific gene transfer vectors are known in the art.

[0063] One consideration associated with commercial exploitation oftransgenic plants is resistance management. This is of particularconcern with Bacillus thuringiensis toxins. There are numerous companiescommercially exploiting Bacillus thuringiensis and there has been muchconcern about development of resistance to Bt toxins. One strategy forinsect resistance management would be to combine the toxins produced byXenorhabdus with toxins such as Bt, vegetative insecticidal proteinsfrom Bacillus stains (Ciba Geigy; WO 94/21795) or other insect toxins.The combinations could be formulated for a sprayable application orcould be molecular combinations. Plants could be transformed withXenorhabdus genes that produce insect toxins and other insect toxingenes such as Bt.

[0064] European Patent Application 040024GAl describes transformation ofa plant with 2 Bts. This could be any 2 genes, not just Bt genes.Another way to produce a transgenic plant that contains more than oneinsect resistant gene would be to produce two plants, with each plantcontaining an insect resistance gene. These plants could then bebackcrossed using traditional plant breeding techniques to produce aplant containing more than one insect resistance gene.

[0065] In addition to producing a transformed plant, there are otherdelivery systems where it may be desirable to re-engineer the bacterialgene(s). Along the same lines, a genetically engineered, easily isolatedprotein toxin made by fusing together both a molecule attractive toinsects as a food source and the functional activity of the toxin may beengineered and expressed in bacteria or in eukaryotic cells usingstandard, well-known techniques. After purification in the laboratorysuch a toxic agent with “built-in” bait could be packaged insidestandard insect trap housings.

[0066] Another delivery scheme is the incorporation of the geneticmaterial of toxins into a baculovirus vector. Baculoviruses infectparticular insect hosts, including those desirably targeted with theXenorhabdus toxins. Infectious baculovirus harboring an expressionconstruct for the Xenorhabdus toxins could be introduced into areas ofinsect infestation to thereby intoxicate or poison infected insects.

[0067] Insect viruses, or baculoviruses, are known to infect andadversely affect certain insects. The affect of the viruses on insectsis slow, and viruses do not immediately stop the feeding of insects.Thus, viruses are not viewed as being optimal as insect pest controlagents. However, combining the Xenorhabdus toxin genes into abaculovirus vector could provide an efficient way of transmitting thetoxins. In addition, since different baculoviruses are specific todifferent insects, it may be possible to use a particular toxin toselectively target particularly damaging insect pests. A particularlyuseful vector for the toxins genes is the nuclear polyhedrosis virus.Transfer vectors using this virus have been described and are now thevectors of choice for transferring foreign genes into insects. Thevirus-toxin gene recombinant may be constructed in an orallytransmissible form. Baculoviruses normally infect insect victims throughthe mid-gut intestinal mucosa. The toxin gene inserted behind a strongviral coat protein promoter would be expressed and should rapidly killthe infected insect.

[0068] In addition to an insect virus or baculovirus or transgenic plantdelivery system for the protein toxins of the present invention, theproteins may be encapsulated using Bacillus thuringiensis encapsulationtechnology such as but not limited to U.S. Patent Nos. 4,695,455;4,695,462; 4,861,595 which are all incorporated herein by reference.Another delivery system for the protein toxins of the present inventionis formulation of the protein into a bait matrix, which could then beused in above and below ground insect bait stations. Examples of suchtechnology include but are not limited to PCT Patent Application WO93/23998, which is incorporated herein by reference.

[0069] Plant RNA viral based systems can also be used to expressXenorhabdus toxin. In so doing, the gene encoding a Xenorhabdus toxincan be inserted into the coat promoter region of a suitable plant viruswhich will infect the host plant of interest. The Xenorhabdus toxin canthen be expressed thus providing protection of the plant from insectdamage. Plant RNA viral based systems are described in U.S. Pat No.5,500,360 to Mycgoen Plant Sciences, Inc. and U.S. Pat Nos. 5,316,931and 5,589,367 to Biosource Genetics Corp. which are incorporated hereinby reference.

[0070] Standard and molecular biology techniques may be used to cloneand sequence the toxins described herein. Additional information may befound in Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989),Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, whichis incorporated herein by reference.

[0071] The following abbreviations are used throughout the Examples:Tris=tris (hydroxymethyl) amino methane; SDS=sodium dodecyl sulfate;EDTA=ethylenediaminetetraacetic acid, IPTG=isopropylthio-B-galactoside,X-gal=5-bromo-4-chloro-3-indoyl-B-D-galactoside,CTAB=cetyltrimethylammonium bromide; kbp=kilobase pairs; DATP, dCTP,dGTP, dTTP, I=2′-deoxynucleoside 5′-triphosphates of adenine, cytosine,guanine, thymine, and inosine, respectively; ATP=adenosine 5′triphosphate.

[0072] The particular embodiments of this invention are furtherexemplified in the Examples. However, those skilled in the art willreadily appreciate that the specific experiments detailed are onlyillustrative of the invention as described more fully in the claimswhich follow thereafter.

EXAMPLE 1 Characterization of Xenorhabdus Strains

[0073] In order to establish that the collection described hereinconsisted of Xenorhabdus isolates, strains were assessed in terms ofrecognized microbiological traits that are characteristic of phase Ivariants of Xenorhabdus and which differentiate it from otherEnterobacteriaceae and Photorhabdus spp. [Farmer, J. J. 1984. Bergey'sManual of Systemic Bacteriology, vol 1. pp. 510-511. (ed. Kreig N. R.and Holt, J. G.). Williams & Wilkins, Baltimore.; Akhurst and Boemare,1988, J. Gen. Microbiol. 134, 1835-1845; Forst and Nealson, 1996.Microbiol. Rev. 60, 21-43]. These characteristic traits were as follows:Gram stain negative rods; organism size of 0.3-2 μm in width and 2-10 μmin length with occasional filaments (15-50 μm) and spheroplasts; whiteto yellow/brown colony pigmentation on nutrient agar; presence ofcrystalline inclusion bodies; absence of catalase; negative for oxidase;inability to reduce nitrate; absence of bioluminescence; ability to takeup dye from growth media; positive for protease production;growth-temperature below 37° C.; survival under anaerobic conditions andpositively motile (Table 3). Methods were checked using referenceEscherichia coli, Xenorhabdus and Photorhabdus strains as controls.Overall results shown in Table 3 were consistent with all strains beingmembers of the family Enterobacteriaceae and the genus Xenorhabdus.

[0074] A luminometer was used to establish the absence ofbioluminescence associated with Xenorhabdus strains. To measure thepresence or absence of relative light emitting units, broth from eachstrain (cells and media) was measured at up to three time intervalsafter inoculation in liquid culture (24, 48 and/or 72 h) and compared tobackground luminosity (uninoculated media). Several Photorhabdus strainswere also tested as positive controls for luminosity. Prior to measuringlight emission from selected broths, cell density was established bymeasuring A_(560 nm) in a Gilford Systems (Oberlin, Ohio)spectrophotometer using a sipper cell. The resulting light emittingunits were then normalized to cell density. Aliquots of broths wereplaced into 96-well microtiter plates (100 μL each) and read in aPackard Lumicount luminometer (Packard Instrument Co., Meriden Conn.).The integration period for each sample was 0.1 to 1.0 sec. The sampleswere agitated in the luminometer for 10 sec prior to taking readings. Apositive test was determined as being ≧3-fold background luminescence(˜1-5 relative light units). In addition, absence of colony luminositywith some strains was confirmed with photographic film overlays andvisual analysis after visual adaptation in a darkroom.

[0075] The Gram staining characteristics of each strain were establishedwith a commercial Gram-stain kit (BBL, Cockeysville, Md.) in conjunctionwith Gram stain control slides (Fisher Scientific, Pittsburgh, Pa.).Microscopic evaluation was then performed using a Zeiss microscope (CarlZeiss, Germany) 100× oil immersion objective lens (with 10× ocular and2× body magnification). Microscopic examination of individual strainsfor organism size, cellular description and inclusion bodies (the lattertwo observations after logarithmic growth) was performed using wet mountslides (10× ocular, 2× body and 40× objective magnification) and phasecontrast microscopy with a micrometer (Akhurst, R. J. and Boemare, N. E.1990. Entomopathogenic Nematodes in Biological Control (ed. Gaugler, R.and Kaya, H.). pp. 75-90. CRC Press, Boca Raton, USA.; Baghdiguian S.,Boyer-Giglio M. H., Thaler, J. O., Bonnot G., Boemare N. 1993. Biol.Cell 79, 177-185). Colony pigmentation was observed after inoculation onBacto nutrient agar, (Difco Laboratories, Detroit, Mich.) prepared perlabel instructions. Incubation occurred at 28° C. and descriptions wererecorded after 5-7 days.

[0076] To test for the presence of catalase activity, 1 mL of culturebroth or a colony of the test organism on a small plug of nutrient agarwas placed into a glass test tube. One mL of a household hydrogenperoxide solution was gently added down the side of the tube. A positivereaction was recorded when bubbles of gas (presumably oxygen) appearedimmediately or within 5 sec. Negative controls of uninoculated nutrientagar or culture broth and hydrogen peroxide solution were also examined.

[0077] Theoxidase reaction of each strain was determined by rubbing 24 hcolonies onto DrySlide Oxidase slides (Difco, Inc.; Detroit, Mich.).Oxidase positive strains produce a dark purple color, indicative ofcytochrome oxidase C, within 20 sec after the organism was rubbedagainst the slide. Failure to produce a dark purple color indicated thatthe organism was oxidase negative.

[0078] To test for nitrate reduction, each culture was inoculated into10 mL of Bacto Nitrate Broth (Difco Laboratories, Detroit, Mich.). After24 h incubation at 28° C., nitrite production was tested by the additionof two drops of sulfanilic acid reagent and two drops ofalpha-naphthylamine reagent (Difco Manual, 10th edition, DifcoLaboratories, Detroit, Mich., 1984). The generation of a distinct pinkor red color indicated the formation of nitrite from nitrate whereas thelack of color formation indicated that the strain was nitrate reductionnegative. In the latter case, finely powdered zinc was added to furtherconfirm the presence of unreduced nitrate established by the formationof nitrite and the resultant red color.

[0079] The ability of each strain to uptake dye from growth media wastested with Bacto MacConkey agar containing the dye neutral red; BactoTergitol-7 agar containing the dye bromothymol blue and Bacto EMB Agarcontaining the dyes methylene blue and eosin-Y (formulated agars fromDifco Laboratories, Detroit, Mich., all prepared according to labelinstructions). After inoculation on these media, dye uptake was recordedupon incubation at 28° C. for 5 days. Growth on Bacto MacConkey andBacto Tergitol-7 media is characteristic for members of the familyEnterobacteriaceae. Motility of each strain was tested using a solutionof Bacto Motility Test Medium (Difco Laboratories, Detroit, Mich.)prepared per label instructions. A butt-stab inoculation was performedwith each strain and positive motility was judged after incubation at28° C. by macroscopic observation of a diffuse zone of growth spreadingfrom the line of inoculation.

[0080] The production of protease was tested by observing hydrolysis ofgelatin using Bacto gelatin (Difco Laboratories, Detroit, Mich.) platesmade per label instructions. Cultures were inoculated and the plateswere incubated at 22° C. for 3-5 days prior to assessment of gelatinhydrolysis. To assess growth at different temperatures, agar plates [2%proteose peptone #3 with two percent Bacto-Agar (Difco, Detroit, Mich.)in deionized water] were streaked from a common source of inoculum.Plates were incubated at 20, 28 and 37° C. for 5 days. The incubatortemperatures were checked with an electronic thermocouple and metered toinsure valid temperature settings.

[0081] Oxygen requirements for Xenorhabdus strains were tested in thefollowing manner. A butt-stab inoculation into fluid thioglycolate brothmedium (Difco, Detroit, Mich.) was made. The tubes were incubated atroom temperature for one week and cultures were then examined for typeand extent of growth. The indicator resazurin was used to indicate thepresence of medium oxygenation or the aerobiosis zone (Difco Manual,10th edition, Difco Laboratories, Detroit, Mich.). In the case ofunclear results, the final agar concentration of fluid thioglycolatebroth medium was raised to 0.75% and the growth characteristicsrechecked.

[0082] The diversity of Xenorhabdus strains was measured by analysis ofPCR (Polymerase Chain Reaction) mediated genomic fingerprinting usinggenomic DNA from each strain. This technique is based on families ofrepetitive DNA sequences present throughout the genome of diversebacterial species (reviewed by Versalovic, J., Schneider, M., D EBruijn, F. J. and Lupski, J. R. 1994. Methods Mol. Cell. Biol., 5,25-40). Three of these, repetitive extragenic palindromic sequence(REP), enterobacterial repetitive intergenic consensus (ERIC) and theBOX element, are thought to play an important role in the organizationof the bacterial genome. Genomic organization is believed to be shapedby selection and the differential dispersion of these elements withinthe genome of closely related bacterial strains can be used todiscriminate between strains (e.g. Louws, F. J., Fulbright, D. W.,Stephens, C. T. and D E Bruijn, F. J. 1994. Appl. Environ. Micro. 60,2286-2295). Rep-PCR utilizes oligonucleotide primers complementary tothese repetitive sequences to amplify the variably sized DNA fragmentslying between them. The resulting products are separated byelectrophoresis to establish the DNA “fingerprint” for each strain.

[0083] To isolate genomic DNA from strains, cell pellets wereresuspended in TE buffer (10 or 50 mM Tris-HCl, 1 or 50 mM EDTA, pH 8.0)to a final volume of 10 mL and 12 mL of 5 M NaCl was then added. Thismixture was centrifuged 20 min at 15,000 ×g. The resulting pellet wasresuspended in 5.7 mL of TE and 300 μL of 10% SDS and 60 μL 20 mg/mlproteinase K (Gibco BRL Products, Grand Island, N.Y.) were added. Thismixture was incubated at 37° C. for 1 h, about 10 mg of lysozyme wasadded, and the mixture was then incubated for an additional 30 to 45min. One mL of SM NaCl and 800 μL of CTAB/NaCl solution (10% w/v CTAB,0.7 M NaCl) were then added and the mixture was incubated 10 to 20 minat 65° C., and in some cases, gently agitated, then incubated andagitated for an additional 20 min to aid in clearing of the cellularmaterial. An equal volume of chloroform/isoamyl alcohol solution ( 24:1,v/v) was added, mixed gently then centrifuged. Two extractions wereperformed with an equal volume of phenol/chloroform/isoamyl alcohol(PCI; 50:49:1). Genomic DNA was precipitated with 0.6 volume ofisopropanol. Precipitated DNA was removed with a sterile plastic loop orglass rod, washed twice with 70% ethanol, dried TABLE 3 Taxonomic Traitsof Xenorhabdus Strains Strain A* B C D E F G H I J^(§) K L M N O P Q RS. carp −^(') + − rd S + − − + + W + + + + + + − − X. Wi − + − rd S + −− + + W + + + + + + − − X. nem − + − rd S + − − + + W + + + + + + − − X.NH3 − + − rd S + − − + + W + + + + + + − − X. riobravis − + − rd S + −− + + W + + + + + + − − DEX1 − + − rd S + − − + + W + + + + + + − − DEX6− + − rd S + − − + + W + + + + + + − − ILM037 − + − rd S + − − + +C + + + + + + − − ILM039 − + − rd S + − − + + W + + + + + + − − ILM070− + − rd S + − − + + W + + + + + + − − ILM078 − + − rd S + − − + +W + + + + + + − − ILM079 − + − rd S + − − + + C + + + + + + − − ILM080− + − rd S + − − + + W + + + + + + − − ILM081 − + − rd S + − − + +W + + + + + + − − ILM082 − + − rd S + − − + + W + + + + + + − − ILM083− + − rd S + − − + + W + + + + + + − − ILM084 − + − rd S + − − + +W + + + + + + − − ILM102 − + − rd S + − − + + C + + + + + + − − ILM103− + − rd S + − − + + C + + + + + + − − ILM104 − + − rd S + − − + +C + + + + + + − − ILM129 − + − rd S + − − + + Y + + + + + + − − ILM133− + − rd S + − − + + Y + + + + + + − − ILM135 − + − rd S + − − + +Y + + + + + + − − ILM138 − + − rd S + − − + + Y + + + + + + − − ILM142− + − rd S + − − + + Y + + + + + + − − ILM143 − + − rd S + − − + +Y + + + + + + − − GLX26 − + − rd S + − − + + C + + + + + + − − GLX40 − +− rd S + − − + + C + + + + + + − − GLX166 − + − rd S + − − + +C + + + + + + − − SEX20 − + − rd S + − − + + C + + + + + + − − SEX76 − +− rd S + − − + + C + + + + + + − − SEX180 − + − rd S + − − + +C + + + + + + − − GL133B − + − rd S + − − + + Y + + + + + + − − DEX2 − +− rd S + − − + + W + + + + + + − − DEX3 − + − rd S + − − + +Y + + + + + + − − DEX4 − + − rd S + − − + + W + + + + + + − − DEX5 − + −rd S + − − + + W + + + + + + − − DEX7 − + − rd S + − − + + W + + + + + +ND − DEX8 − + − rd S + − − + + W + + + + + + ND −

[0084] and dissolved in 2 mL of STE (10 mM Tris-HCl pH8.0, 10 mM NaCl, 1mM EDTA). The DNA was then quantitated at A_(260 nm). In a secondmethod, 0.01 volumes of RNAase A (50 μg/mL final) was added andincubated at 37° C. for 2 h. The sample was then extracted with an equalvolume of PCI. The samples were then precipitated with 2 volumes of 100%ethanol and collected as described above. Samples were then air driedand resuspended in 250-1000 μL of TE.

[0085] To perform rep-PCR analysis of Xenorhabdus genomic DNA, thefollowing primers were used: REP1R-I; 5′-IIIICGICGICATCIGGC-3′ andREP2-I; 5′-ICGICTTATCIGGCCTAC-3′. PCR was performed using the following25 μL reaction: 7.75 μL H₂O, 2.5 μL 10× LA buffer (PanVera Corp.,Madison, Wis.), 16 μL DNTP mix (2.5 mM each), 1 μL of each primer at 50pM/μL, 1 μL DMSO, 1.5 μL genomic DNA (concentrations ranged from0.075-0.480 μg/μL) and 0.25 μL TaKaRa EX Taq (PanVera Corp., Madison,Wis.). The PCR amplification was performed in a Perkin Elmer DNA ThermalCycler (Norwalk, Conn.) using the following conditions: 95° C. for 7 minthen [94° C. for 1 min, 44° C. for 1 min, 65° C. for 8 min] for 35cycles; followed by 65° C. for 15 min. After cycling, 25 μL of reactionwas added to 5 μL of 6× gel loading buffer (0.25% bromophenol blue, 40%w/v sucrose in H₂O). A 15×20 cm 1%-agarose gel was then run in TBEbuffer (0.09 M Tris-borate, 0.002 M EDTA) using 8 μL of each reaction.The gel was run for approximately 16 h at 45 V. Gels were then stainedin 20 μg/mL ethidium bromide for 1 h and destained in TBE buffer forapproximately 3 h. Polaroid® photographs of the gels were then takenunder UV illumination.

[0086] The presence or absence of bands at specific sizes for eachstrain was scored from the photographs using RFLP scan Plus software(Scanalytics, Billerica, Mass.) and entered as a similarity matrix inthe numerical taxonomy software program, NTSYS-pc (Exeter Software,Setauket, N.Y.). Controls of E. coli strain HE101 and Xanthomonas oryzaepv. oryzae assayed under the same conditions produced PCR fingerprintscorresponding to published reports (Versalovic, J., Koeuth, T. andLupski, J. R. 1991. Nucleic Acids Res. 19, 6823-6831; Vera Cruz, C. M.,Halda-Alija, L., Louws, F., Skinner, D. Z., George, M. L., Nelson, R.J., D E Bruijn, F. J., Rice, C. and Leach, J. E. 1995. Int. Rice Res.Notes, 20, 23-24.; Vera Cruz, C. M., Ardales, E. Y., Skinner, D. Z.,Talag, J., Nelson, R. J., Louws, F. J., Leung, H., Mew, T. W. and Leach,J. E. 1996. Phytopathology 86, 1352-1359). The data from Xenorhabdusstrains were then analyzed with a series of programs within NTSYS-pc;SIMQUAL (Similarity for Qualitative data) to generate a matrix ofsimilarity coefficients (using the Jaccard coefficient) and SAHN(Sequential, Agglomerative, Heirarchical and Nested) clustering usingthe UPGMA method (Unweighted Pair-Group Method with Arithmetic Averages)which groups related strains and can be expressed as a phenogram (FIG.1). The COPH (cophenetic values) and MXCOMP (matrix comparison) programswere used to generate a cophenetic value matrix and compare thecorrelation between this and the original matrix upon which theclustering was based. A resulting normalized Mantel statistic (r) wasgenerated which was a measure of the goodness of fit for a clusteranalysis (r=0.8-0.9 representing a very good fit). In our case r=0.9,indicated an excellent fit. Therefore, strains disclosed herein weredetermined to be comprised of a diverse group of easily distinguishablestrains representative of the Xenorhabdus genus.

[0087] Strains disclosed herein were deposited before application filingwith the following International Deposit Authority: AgriculturalResearch Service Patent Culture Collection (NRRL), National Center forAgricultural Utilization Research, ARS-USDA, 1815 North University St.,Peoria, Ill. 61604. The following strains , with NRRL designations weredeposited Apr. 29, 1997: S. Carp (NRRL-B-21732); X. Wi (NRRL-B-21733);X. nem (NRRL-B-21734); X. NH3 (NRRL-B-21735); X. riobravis(NRRL-B-21736); GL 133B (NRRL-B-21737); DEX1 (NRRL-B-21738); DEX2(NRRL-B-21739); DEX3 (NRRL-B-21740); DEX4 (NRRL-B-21741); DEX 5(NRRL-B-21742); and DEX 6 (NRRL-B-21743). The remaining strainsdisclosed herein were deposited with NRRL on Apr. 30, 1998. In all,thirty-nine (39) strains were deposited.

EXAMPLE 2 Functional Utility of Toxin(S) Produced by Various XenorhabdusStrains

[0088] “Storage” cultures of the various Xenorhabdus strains wereproduced by inoculating 175 mL of 2% Proteose Peptone #3 (PP3) (DifcoLaboratories, Detroit, Mich.) liquid medium with a phase I variantcolony in a 500 mnL tribaffled flask with a Delong neck covered with aKaput closure. After inoculation, flasks were incubated for between24-72 h at 28° C. on a rotary shaker at 150 rpm. Cultures were thentransferred to a sterile bottle containing a sterile magnetic stir barand then over-layered with sterile mineral oil to limit exposure to air.Storage cultures were kept in the dark at room temperature. Thesecultures were then used as inoculum sources for the fermentation of eachstrain. Phase I variant colonies were also stored frozen at −70° C. foruse as an inoculum source. Single, phase I colonies were selected fromPP3 plates containing bromothymol blue (0.0025%) and placed in 3.0 mLPP3 and grown overnight on a rotary shaker (150 rpm) at 28° C. Glycerol(diluted in PP3) was then added to achieve a final concentration of 20%and the cultures were frozen in aliquots at −70° C. For cultureinoculation, a portion of the frozen aliquot was removed aseptically andstreaked on PP3 containing bromothymol blue for reselection of phase Icolonies.

[0089] Pre-production “seed” flasks or cultures were produced by eitherinoculating 2 mL of an oil over-layered storage culture or bytransferring a phase I variant colony into 175 mLt sterile medium in a500 mL tribaffled flask covered with a Kaput closure. Typically,following 16 h incubation at 28° C. on a rotary shaker at 150 rpm, seedcultures were transferred into production flasks. Production flasks wereusually inoculated by adding ˜1% of the actively growing seed culture tosterile PP3 or tryptic soy broth (TSB, Difco Laboratories, DetroitMich.). For small-scale productions, flasks were inoculated directlywith a phase I variant colony. Production of broths occurred in 500 mLtribaffled flasks covered with a Kaput closure. Production flasks wereagitated at 28° C. on a rotary shaker at 150 rpm. Productionfermentations were terminated after 24-72 h.

[0090] Following appropriate incubation, broths were dispensed intosterile 1.0 L polyethylene bottles, spun at 2600 ×g for 1 h at 10° C.and decanted from the cell and debris pellet. Broths were then filtersterilized or further broth clarification was achieved with a tangentialflow microfiltration device (Pall Filtron, Northborough, Mass.) using a0.5 μM open-channel poly-ether sulfone (PES) membrane filter. Theresulting broths were then concentrated (up to 10-fold) using a 10,000or 100,000 MW cut-off membrane, M12 ultra-filtration device (Amicon,Beverly Mass.) or centrifugal concentrators (Millipore, Bedford, Mass.and Pall Filtron, Northborough, Mass.) with a 10,000 or 100,000 MW poresize. In the case of centrifugal concentrators, broths were spun at 2000×g for approximately 2 h. The membrane permeate was added to thecorresponding retentate to achieve the desired concentration ofcomponents greater than the pore size used. Following these procedures,broths were used for biochemical analysis or biological assessment. Heatinactivation of processed broth samples was achieved by heating 1 mLsamples at 100° C. in a sand-filled heat block for 10-20 min.

[0091] Broth(s) and toxin complex(es) from different Xenorhabdus strainswere useful for reducing populations of insects and were used in amethod of inhibiting an insect population which comprised applying to alocus of the insect an effective insect inactivating amount of theactive described. A demonstration of the breadth of functional activityobserved from broths of a selected group of Xenorhabdus strainsfermented as described above is shown in Table 4. It is possible thatimproved or additional functional activities could be detected withthese strains through increased concentration of the broth or byemploying different fermentation methods as disclosed herein. Consistentwith the activity being associated with a protein, the functionalactivity showed heat lability and/or was present in the high molecularweight retentate (greater than 10 kDa and predominantly greater than 100kDa) after concentration of the broth.

[0092] Culture broth(s) from diverse Xenorhabdus strains showeddifferential functional activity (mortality and/or growth inhibition)against a number of insects. More specifically, activity was seenagainst corn rootworm larvae and boll weevil larvae which are members ofthe insect order Coleoptera. Other members of the Coleoptera includewireworms, pollen beetles, flea beetles, seed beetles and Coloradopotato beetle. The broths and purified toxin complex(es) were alsoactive against tobacco budworm, tobacco hornworm, corn earworm andEuropean corn borer which are members of the order Lepidoptera. Othertypical members of this order are beet armyworm, cabbage looper, blackcutworm, codling moth, clothes moth, Indian mealmoth, leaf rollers,cabbage worm, bagworm, Eastern tent caterpillar, sod webworm and fallarmyworm. Activity was also seen against mosquito larvae which aremembers of the order Diptera. Other members of the order Diptera are,pea midge, carrot fly, cabbage root fly, turnip root fly, onion fly,crane fly and house fly and various mosquito species. Activity withbroth(s) was also seen against two-spotted spider mite which is a memberof the order Acarina which includes strawberry spider mites, broadmites, citrus red mite, European red mite, pear rust mite and tomatorusset mite.

[0093] Activity against corn rootworm larvae was tested as follows.Xenorhabdus culture broth(s) (10× concentrated, filter sterilized), PP3or TSB (10× concentrated), purified toxin complex(es) or 10 mM sodiumphosphate buffer , pH 7.0, were applied directly to the surface (about1.5 cm²) of artificial diet (Rose, R. I. and McCabe, J. M. 1973. J.Econ. Entomol. 66, 398-400) in 40 μL aliquots. Toxin complex was dilutedin 10 mM sodium phosphate buffer, pH 7.0. The diet plates were allowedto air-dry in a sterile flow-hood and the wells were infested withsingle, neonate Diabrotica undecimpunctata howardi (Southern cornrootworm, SCR) hatched from surface sterilized eggs. Plates were sealed,placed in a humidified growth chamber and maintained at 27° C. for theappropriate period (3-5 days). Mortality and larval weightdeterminations were then scored. Generally, 8-16 insects per treatmentwere used in all studies. Control mortality was generally less than 5%.

[0094] Activity against boll weevil (Anthomonas grandis) was tested asfollows. Concentrated (10×) Xenorhabdus broths or control medium (PP3)were applied in 60 μL aliquots to the surface of 0.35 g of artificialdiet (Stoneville Yellow lepidopteran diet) and allowed to dry. A single,12-24 h boll weevil larva was placed on the diet, the wells were sealedand held at 25° C., 50% relative humidity (RH) for 5 days. Mortality andlarval weights were then assessed. Control mortality ranged between0-25%.

[0095] Activity against mosquito larvae was tested as follows. The assaywas conducted in a 96-well microtiter plate. Each well contained 200 μLof aqueous solution (10×concentrated Xenorhabdus culture broth(s),control medium (2% PP3) and about 20, 1-day old larvae (Aedes aegypti).There were 6 wells per treatment. The results were read at 24 h afterinfestation. No control mortality was observed.

[0096] Activity against lepidopteran larvae was tested as follows.Concentrated (1×) Xenorhabdus culture broth(s), control medium (PP3 orTSB), purified toxin complex(es) or 10 mM sodium phosphate buffer, pH7.0 were applied directly to the surface (˜1.5 cm²) of standardartificial lepidopteran diet (Stoneville Yellow diet) in 40 μL aliquots.The diet plates were allowed to air-dry in a sterile flow-hood and eachwell was infested with a single, neonate larva. European corn borer(Ostrinia nubilalis), fall armyworm (Spodoptera frugiperda), cornearworm (Helicoverpa zea) and tobacco hornworm (Manduca sexta) eggs wereobtained from commercial sources and hatched in-house whereas tobaccobudworm (Heliothis virescens) and beet armyworm (Spodoptera exigua)larvae were supplied internally. Following infestation with larvae, dietplates were sealed, placed in a humidified growth chamber and maintainedin the dark at 27° C. for the appropriate period. Mortality and weightdeterminations were scored at day 5. Generally, 16 insects per treatmentwere used in all studies. Control mortality generally ranged from0-12.5%.

[0097] Activity against two-spotted spider mite (Tetranychus urticae)was determined as follows. Young squash plants were trimmed to a singlecotyledon and sprayed to run-off with 10× concentrated broth(s) orcontrol medium (PP3). After drying, plants were infested with a mixedpopulation of spider mites and held at room temperature and humidity for72 hr. Live mites were then counted to determine levels of control.

EXAMPLE 3 Functional Activity of Highly Purified Toxin Proteins fromXenorhabdus Strain X. riobravis

[0098] Functional toxin protein was purified from fermentation broth ofXenorhabdus strain X. riobravis as described herein. This toxin wastested against neonate larvae of five insect species, Southern cornrootworm, European cornborer, Tobacco hornworm, Corn earworm and Tobaccobudworm following the methods described in Example 2. The results areseen in Table 5. All species showed growth inhibitory and/or lethaleffects after five days when presented with toxin at a dose of 440 ngtoxin/cm² diet. TABLE 4 Observed Functional Spectrum of Broths FromDifferent Xenorhabdus Strains Xenorhabdus strain Sensitive* InsectSpecies S. carp 1**, 2, 3, 4, 5, 6, 7 X. riobravis 1, 2, 3, 5, 6, 7 X.NH3 1, 2, 3, 6 X. Wi 1, 2, 3, 5, 6, 7 X. nem 3, 5, 6 DEX1 1, 2, 3, 6DEX6 1, 2, 3, 4, 5, 6 ILM037 1, 4 ILM039 4 ILM070 4, 8 ILM078 3, 4ILM079 3 ILM080 3 ILM081 3 ILM082 3 ILM083 3 ILM084 3 ILM102 1, 2, 4ILM103 1, 3, 4, 8 ILM104 3, 4, 8 ILM116 1, 4 ILM129 1, 4 ILM133 1, 4ILM135 1, 2, 4 ILM138 4 ILM142 1, 2, 3, 4, 8 ILM143 4 GLX26 8 GLX40 3, 8GLX166 4 SEX20 1, 4, 8 SEX76 1, 4 SEX180 4 GL 133B 4 DEX2 6, 7 DEX3 3, 6DEX4 6, 7 DEX5 3, 6 DEX7 3 DEX8 3

[0099] TABLE 5 Effect of Highly Purified X. riobravis Toxin on VariousInsect Species S. corn European Tobacco Corn Tobacco Treatment rootwormcornborer hornworm earworm budworm X. 19/46* 75/61 75/75 25/95 13/98riobravis

EXAMPLE 4 Effect of Different Culture Media on Functional Activity ofFermentation Broths from Selected Xenorhabdus Strains

[0100] Several different culture media were used to further optimizeconditions for detection of functional activity in the fermentationbroths of several Xenorhabdus strains. GL133B, X. riobravis, X. Wi, DEX8and DEX1 were grown in PP3, TSB and PP3 plus 1.25% NaCl (PP3S) asdescribed herein. Broths were then prepared as described herein andassayed against neonate Tobacco hornworm to determine any changes ininsecticidal activity. In both experimental cases (condition A which isPP3 vs. TSB; and condition B which is PP3 vs. PP3S), the functionalactivity of fermentations in PP3S and/or TSB were improved as comparedto simultaneous PP3 fermentations (Table 6). In certain cases, activitywas uncovered which was not apparent with PP3 fermentations. Thefunctional activity produced under condition A and condition B was shownto be heat labile and retained by high molecular weight membranes(>100,000 kDa). Addition of NaCl to broth after bacterial growth wascomplete did not increase toxin activity indicating that the increasedfunctional activity observed was not due to increase NaCl concentrationin the media but instead due to increased toxin.

[0101] The increased activity observed with X. riobravis fermented inPP3S was further investigated by partial purification of toxin(s) fromfermentations in PP3 and PP3S as described herein. Consistent withobservations using culture broth, the active fraction(s) from PP3S broth(obtained from anion exchange and size-exclusion chromatography asdescribed herein) contained increased biological activity, proteinconcentration and a more complex protein pattern as determined bySDS-PAGE analysis. TABLE 6 The Effect of Different Culture Media onFunctional Potency of Selected Xenorhabdus Fermentation Broths ConditionA Condition B Strains PP3 TSB PP3 PP3S GL133B −* − − + X. riobravis ++++ + +++ X. Wi + +++ + +++ DEX8 − + − − DEX6 + ++ + +++ Control − − − −

EXAMPLE 5 Xenorhabdus Strains X.nem, X. riobravis, and X. Wi:Purification, Characterization and Activity

[0102] The protocol, as follows, was established based on purifyingthose fractions having the most activity against EYE Tobacco Hornworm(Manduca sexta) , hereinafter THW, as determined in bioassays (seeExample 2). Typically, 4-20 L of Xenorhabdus culture that had been grownin PP3 broth being filtered, as described herein, were received andconcentrated using an Amicon spiral ultra filtration cartridge TypeSlYlGO attached to an Amicon M-12 filtration device (Amicon Inc.,Beverly, Mass.). The retentate contained native proteins wherein themajority consisted of those having molecular sizes greater than 100 kDa,whereas the flow through material contained native proteins less than100 kDa in size. The majority of the activity against THW was containedin the 100 kDa retentate. The retentate was then continually diafilteredwith 10 mM sodium phosphate (pH=7.0) until the filtrate reached an A₂₈₀<0.100. Unless otherwise stated, all procedures from this point wereperformed in buffer defined as 10 mM sodium phosphate (pH 7.0). Theretentate was then concentrated to a final volume of about 0.20 L andthen filtered using a 0.45 μm sterile filtration unit (Corning, Corning,N.Y.).

[0103] The filtered material was loaded at 7.5 mL/min onto a PharmaciaHR16/10 column which had been packed with PerSeptive Biosystem POROS 50HQ strong anion exchange matrix equilibrated in buffer using aPerSeptive Biosystem SPRINT HPLC system (PerSeptive Biosystems,Framingham, Mass.). After loading, the column was washed with bufferuntil an A_(280 nm) <0.100 was achieved. Proteins were then eluted fromthe column at 2.5 mL/min using buffer with 0.4 M NaCl for 20 min for atotal volume of 50 mL. The column was then washed using buffer with 1.0M NaCl at the same flow rate for an additional 20 min (final volume=50ml). Proteins eluted with 0.4 M and 1.0 M NaCl were placed in separatedialysis bags (SPECTRA/POR Membrane MWCO: 2,000; Spectrum, Houston,Tex.) and allowed to dialyze overnight at 4° C. in 12 L buffer. In somecases, the 0.4 M fraction was not dialyzed but instead was immediatelydesalted by gel filtration (see below). The majority of activity againstTHW was contained in the 0.4 M fraction.

[0104] The 0.4 M fraction was further purified by application of 20 mLto a Pharmacia XK 26/100 column that had been prepacked with SepharoseCL4B (Pharmacia) using a flow rate of 0.75 mL/min. Fractionation of the0.4 M fraction on the Sepharose CL4B column yielded four to fivedistinct peaks when purifying X. nem and X. Wi. Proteins from strain X.riobravis, while having a distinct peak equivalent to the void volume,also had a very broad, low absorbance region ranging from ca. 280 min toca. 448 min of the 800 min run. Typically, two larger absorbance peakswere observed after 450 min and before 800 min. Active fractions from X.Wi and X. nem typically eluted at about 256 min to 416 min of a 800 minrun

[0105] Fractions were pooled based on A_(280 nm) peak profile andconcentrated to a final volume of 0.75 ml using a Millipore ULTRAFREE-15centrifugal filter device Biomax-50K NMWL membrane (Millipore Inc.,Bedford, Mass.) or concentrated by binding to a Pharmacia MonoQ HR10/10column, as described herein. Protein concentrations were determinedusing a BioRad Protein Assay Kit (BioRad, Hercules, Calif.) with bovinegamma globulin as a standard.

[0106] The native molecular weight of the THW toxin complex wasdetermined using a Pharmacia HR 16/50 column that had been prepackedwith Sepharose CL4B in said phosphate buffer. The column was thencalibrated using proteins of known molecular size thereby allowing forcalculation of the toxin complex approximate native molecular size. Asshown in Table 7, the molecular size of the toxin complex were asfollows: 1500±530 kDa for strain X. nem; 1000±350 kDa for strain X.riobravis; 3290 kDa+1150 kDa for strain X. Wi; 980±245 for strainILM078; 1013±185 for strain DEX6; and 956±307 for strain ILM080. Ahighly purified fraction of X. Wi, said fraction being purified via ionexchange, gel filtration, ion exchange, hydrophobic interactionchromatography, and ion exchange chromatography as disclosed herein wasthen analyzed for size using quantitative gel filtration. This materialwas found to have a native molecular size of 1049±402 kDa (Table 7).

[0107] Proteins found in the toxin complex were examined for individualpolypeptide size using SDS-PAGE analysis. Typically, 20 μg protein ofthe toxin complex from each strain was loaded onto a 2-15%polyacrylamide gel (Integrated Separation Systems, Natick, Mass.) andelectrophoresed at 20 mA in SDS-PAGE buffer (BioRad). After completionof electrophoresis, the gels were stained overnight in BioRad Coomassieblue R-250 (0.2% in methanol: acetic acid: water; 40:10:40 v/v/v).Subsequently, gels were destained in methanol:acetic acid: water;40:10:40 (v/v/v). Gels were then rinsed with water for 15 min andscanned using a Molecular Dynamics PERSONAL LASER DENSITOMETER(Sunnyvale, Calif.). Lanes were quantitated and molecular sizes werecalculated as compared to BioRad high molecular weight standards, whichranged from 200-45 kDa.

[0108] Sizes of individual polypeptides comprising the THW toxin complexfrom each strain are listed in Table 8. The sizes of the individualpolypeptides ranged from 32 kDa to 330 kDa. Each of X. Wi, X. nem, X.riobravis, ILM080, ILM078, and DEX6 strains had polypeptides comprisingthe toxin complex that were in the 160-330 kDa range, the 100-160 kDarange, and the 50-80 kDa range. These data indicate that the toxincomplex may vary in peptide composition and components from strain tostrain; however, in all cases the toxin attributes appears to consist ofa large, oligomeric protein complex with subunits ranging from 23 kDa to330 kDa.

EXAMPLE 5 Sub-fractionation of Xenorhabdus Toxin Complex from X.riobravis and X. Wi

[0109] For subfractionation, about 10 mg of the Xenorhabdus proteintoxin complex of X. riobravis was isolated as described above and wasapplied to a Pharmacia MonoQ HR 10/10 column equilibrated with 10 mMphosphate buffer, pH 7.0 at a flow rate of 2 mL/min. The column waswashed with said buffer until the absorbance at 280 nm returned tobaseline. Proteins bound to the column were eluted with a lineargradient of 0 to 1.0 M NaCl in said buffer at 2 mL/min for 1 h. Two mLfractions were collected and subjected to analysis by bioassay againstTHW as described herein. Peaks of activity were determined by examininga 2-fold dilution of each fraction in THW bioassays. A peak of activityagainst THW was observed that eluted at about 0.3-0.4 M NaCl. Thefractions having activity against THW were pooled and analyzed bySDS-PAGE gel electrophoresis. It was observed that there were fourpredominant peptides having the approximate sizes of 220 kDa, 190 kDa,130 kDa, and 54 kDa.

[0110] The peptides described above were electrophoresed on a 4-20%SDS-PAGE (Integrated Separation Systems) and transblotted to PROBLOTTPVDF membranes (Applied Biosystems, Foster City, Calif.) . Blots weresent for amino acid analysis and N-terminal amino acid sequencing atHarvard MicroChem and Cambridge ProChem, respectively. The aminoterminal sequence of the 220 kDa protein is entered herein as SEQ IDNO:4.

[0111] For sub-fractionation experiments with X. Wi, ca. 10 mg toxin wasapplied to a MonoQ HR 10/10 column equilibrated with 10 mM phosphatebuffer, pH 7.0 at a flow rate of 2 mL/min. The column was washed withsaid buffer until the A_(280 nm) returned to baseline. Proteins bound tothe column were eluted with a linear gradient of 0 to 1.0 M NaCl in saidbuffer at 2 mL/min for 1 h. Two mL fractions were collected andsubjected to analysis by bioassay against THW as described herein. Atleast two major detectable peaks at A_(280 nm) were observed. Themajority of functional THW activity that was observed eluted at about0.10-0.25 M NaCl. The fractions having activity against THW were pooledand analyzed by gel electrophoresis. By SDS-PAGE it was observed thatthere were up to eight predominant peptides having the approximate sizesof 330 kDa, 320 kDa, 270 ka, 220 kDa, 200 kDa, 190 kDa, 170 kDa, 130kla, 91 kDa, 76 kDa, 55 kDa and 36 kDa.

[0112] The peak THW pooled activity fraction was applied tophenyl-sepharose HR 5/5 column. Solid (NH₄)₂SO₄ added to a finalconcentration of 1.7 M. The solution was then applied onto the columnequilibrated with 1.7 M (NH₄)₂SO₄ in 50 mM potassium phosphate buffer,pH 7, at 1 mL/min. Proteins bound to the column were then eluted with alinear gradient of 1.7 M (NH₄)₂SO₄, 50 mM potassium phosphate, pH 7.0 to10 mM potassium phosphate, pH 7.0 at 0.5 mL/min for 60 min. After THWbioassays, it was determined that the peak activity eluted at anA_(280 nm) between 40 min to ca. 50 min. Fractions were dialyzedovernight against 10 mM sodium phosphate buffer, pH 7.0. By SDS-PAGE itwas observed that there were up to six predominant peptides having theapproximate sizes of 270 kDa, 220 kDa, 170 kDa, 130 kDa, and 76 kDa.

[0113] The peptides from THW active fractions from either 5/5 or 10/10phenyl-sepharose column were electrophoresed on a 4-20% SDS-PAGE gel(Integrated Separation Systems) and transblotted to PROBLOTT PVDFmembranes (Applied Biosystems, Foster City, Calif.). Blots were sent foramino acid analysis and N-terminal amino acid sequencing at HarvardMicroChem and Cambridge ProChem, respectively. The N-terminal amino acidsequences for 130 kDa (SEQ ID NO:1), 76 kDa (SEQ ID NO:2), 48 kDa (SEQID NO:5) and 38 kDa (SEQ ID NO:3) peptides are entered herein.

[0114] Insect bioassays were performed using either toxin complex or THWphenyl-sepharose purified fractions. Functional activity (at least 20%mortality) and/or growth inhibition (at least 40%) was observed for fallarmyworm, beet armyworm, tobacco hornworm, tobacco budworm, Europeancorn borer, and southern corn rootworm. In toxin complex preparationstested, higher activity was observed against tobacco hornworm andtobacco budworm than against southern corn rootworm larvae. The insectactivity of X. Wi toxin complex and any additionally purified fractionswere shown to be heat sensitive. TABLE 7 Characterization of a ToxinComplex From Xenorhabdus Strains. STRAIN TOXIN COMPLEX SIZE^(a) X. Wi3290 kDa ± 1150 kDa X. Wi 1049 kDa ± 402 kDa (Highly Purified) X. nem1010 kDa ± 350 kDa X. riobravis 1520 kDa ± 530 kDa ILM 078  980 kDa ±245 kDa ILM 080 1013 kDa ± 185 kDa DEX6  956 kDa ± 307 kDa

[0115] TABLE 8 Molecular Sizes of Peptides in Toxin Complex fromXenorhabdus Strains in kDa. X. Wi X. nem X. riobravis ILM 080 ILM 078DEX 6 330 220 220 200 203 201 320 190 190 197 200 181 270 170 100 173173 148 220 150 96 112 150 138 200 140 92 106 144 128 190 85 85 90 106119 170 79 79 80 80 90 130 65 65 74 62 75 91 56 56 61 58 65 76 50 50 6054 59 55 42 47 58 50 55 49 38 42 55 45 45 46 31 38 53 41 43 29 34 48 3740 26 31 46 32 36 26 43 32 23 42 40

EXAMPLE 6 Production, Isolation, and Characterization of XenorhabdusStrain X. carpocapsae

[0116] A 1% inoculum of an overnight culture of the isolate X.carpocapsae, also known as X. carp, was added to a 125 mL flaskcontaining 25 mL PP3 and incubated for 72 h at 28° C. on a rotary shakerat 250 rpm. Afterwards, the cultures were centrifuged for 20 min at10,000 ×g as described herein followed by filtration of the supernatantusing a 0.2 μm membrane filter. A 15 mL sample of the supernatant wasthen added to an Ultrafree-15 100,000 NMWL centrifugal filter device(Millipore, Mass.) and centrifuged at 2000 ×g. The retentate was washed2× with 100 mM KPO₄, pH 6.9, and then resuspended in 1.0 mL of the same.Proteins were analyzed by SDS-PAGE as disclosed herein using a 10%resolving gel and 4% stacking gel with sizes calibrated using BioRadprestained standards (Hercules, Calif.). Gels were electrophoresed at40V for 16 h at 15° C. and then stained with Colloidal Blue from Novex,Inc., (San Diego, Calif.).

[0117] For additional separations, samples were applied to a BIO-SEPS4000 column (Phenomenex, Torrance, Calif.), 7.5 mm I.D., 60 cm CMLunder an isocratic system using 100 mM KPO₄ pH 6.9. Total amount loadedper sample was 250-500 μg protein. Fractions were collected in 3 groupsdepending on protein size (size exclusion chromatography) as follows:proteins greater than 1,000 kDa; proteins being 800-1,000 kDa; andproteins less than 800,000 kDa. The 800,000˜1,000,000 Da fraction wasselected for further analysis.

[0118] The 800-1000 kDa fractions, which had the most functionalactivity, were pooled and concentrated using a 100,000 NMWL centrifugalfilter devices (Millipore, Bedford, Mass). Each pooled retentatefraction was washed 2× and resuspended in 300 μL of 100 mM KPO₄ pH 6.9.The protein concentrations were determined using the bicinchoninic acidprotein assay reagent kit (Pierce, Rockford, Ill.). Proteins in thisfraction were analyzed by SDS-PAGE as described herein and found to havemany proteins of different sizes. This material was then furtherseparated on a DEAE column whereby proteins were eluted with increasingsalt concentrations. Those fractions having the most activity were thenexamined again via SDS-PAGE and were found to be comprised of 4predominate proteins having sizes as follows: 200, 190, 175 and 45 kDa.The active fraction from the DEAE step was passed through a HPLC gelfiltration column as described above (BioSep S4000) and the toxicactivity against Manduca sexta was found to be contained within afraction having native proteins >800 kDa. Resolution of this fractionvia SDS-PAGE revealed only one protein, said protein having a denaturedsize of 200 kDa. These data suggest that the 200 kDa protein isresponsible for the Manduca sexta functional activity (see below) and ispossibly found as a tetramer in the culture broth.

[0119] Bioassays were performed as follows. Eggs of M. sexta werepurchased from Carolina Biological Supply Co. The eggs were hatched andreared on fresh wheat germ diet (ICN, CA) while incubated at 25° C. in a16 h light/8 h dark photocycle incubator. Oral toxicity data weredetermined by placing twelve M. sexta larva onto a piece of insect foodcontaining 300 μg ultrafiltration retentate obtained as described above.Observations were made over 5 days. For the HPLC-size exclusionchromatography fractions, 20 μg total protein were applied to wheat germdiet. Experiment was repeated in duplicate.

1 5 1 12 PRT Xenorhabdus Wi 1 Asn Gln Asn Val Glu Pro Ser Ala Gly AspIle Val 1 5 10 2 8 PRT Xenorhabdus Wi 2 Ser Gln Asn Val Tyr Arg Tyr Pro1 5 3 7 PRT Xenorhabdus Wi 3 Met Thr Lys Gln Glu Tyr Leu 1 5 4 11 PRTXenorhabdus Wi 4 Met Tyr Ser Thr Ala Val Leu Leu Asn Lys Ile 1 5 10 5 12PRT Xenorhabdus Wi UNSURE (11) 5 Ala Gly Phe Gln Leu Asn Glu Tyr Ser ThrXaa Gly 1 5 10

We claim:
 1. A composition, comprising an effective amount of aXenorhabdus protein toxin having functional activity against an insect,said protein toxin being derived from a protein having a nativemolecular size of at least 100 kDa.
 2. The composition of claim 1,wherein the Xenorhabdus toxin having functional activity against aninsect is produced by a purified culture of Xenorhabdus nematophilus,Xenorhabdus poinarii, Xenorhabdus bovienii, Xenorhabdus beddingii orXenorhabdus species.
 3. The composition of claim 2, wherein saidpurified culture of Xenorhabdus selected from the group consisting of S.carp, X. Wi, X. nem, X. NH3, X. riobravis, GL 133B, DEX1, DEX2, DEX3,DEX4, DEX5, DEX6, DEX7, DEX8, ILM037, ILM039, ILM070, ILM078, ILM079,ILM080, ILM081, ILM082, ILM083, ILM084, ILM102, ILM103, ILM104, ILM129,ILM133, ILM135, ILM138, ILM142, ILM143, GLX26, GLX40, GLX166, SEX20,SEX76, and SEX180.
 4. The composition of claim 1, wherein the toxinhaving functional activity against an insect is produced by a purifiedculture of Xenorhabdus strain designated S. carp, X. Wi, X. nem, X. NH3,X. riobravis, GL 133B, DEX1, DEX2, DEX3, DEX4, DEX5, DEX6, DEX7, DEX8,ILM037, ILM039, ILM070, ILM078, ILM079, ILM080, ILM081, ILM082, ILM083,ILM084, ILM102, ILM103, ILM104, ILM129, ILM133, ILM135, ILM138, ILM142,ILM143, GLX26, GLX40, GLX166, SEX20, SEX76, and SEX180.
 5. Thecomposition of claim 4, wherein the toxin having functional activityagainst an insect is a mixture of one or more toxins produced frompurified cultures of Xenorhabdus.
 6. The composition of claim 3 whereinthe toxin having functional activity against an insect, said toxin beinga mixture of one or more toxins, is produced from said purified culturesof Xenorhabdus, said purified cultures being selected from the groupconsisting of S. carp, X. Wi, X. nem, X. NH3, X. riobravis, GL 133B,DEX1, DEX2, DEX3, DEX4, DEX5, DEX6, DEX7, DEX8, ILM037, ILM039, ILM070,ILM078, ILM079, ILM080, ILM081, ILM082, ILM083, ILM084, ILM102, ILM103,ILM104, ILM129, ILM133, ILM135, ILM138, ILM142, ILM143, GLX26, GLX40,GLX166, SEX20, SEX76, and SEX180.
 7. The composition of claim 1, whereinthe insect is of the order Coleoptera, Lepidoptera, Diptera, or Acarina.8. The composition of claim 7, wherein the insect species from orderColeoptera are selected from the group consisting of Corn Rootworm, BollWeevil, wireworms, pollen beetles, flea beetles, seed beetles andColorado potato beetle.
 9. The composition of claim 7, wherein theinsect species from order Lepidoptera are selected from the groupconsisting of Beet Armyworm, European Corn Borer, Tobacco Hornworm,Tobacco Budworm, cabbage looper, black cutworm, corn earworm, codlingmoth, clothes moth, Indian mealmoth, leaf rollers, cabbage worm, cottonbollworm, bagworm, Eastern tent caterpillar, sod webworm and fallarmyworm.
 10. The composition of claim 7, wherein the insect speciesfrom the order Diptera are selected from the group consisting of peamidge, carrot fly, cabbage root fly, turnip root fly, onion fly, cranefly, house fly, and various mosquito species.
 11. The composition ofclaim 7 wherein the insects species from the order Acarina are selectedfrom the group consisting of two-spotted spider mites, strawberry spidermites, broad mites, citrus red mite, European red mite, pear rust miteand tomato russet mite.
 12. A substantially pure microorganism culturecomprising of Xenorhabdus strain selected from the group consisting ofS. carp, X. Wi, X. nem, X. NH3, X. riobravis, GL 133cn , ILM037, ILM039,ILM070, ILM078, ILM079, ILM080, ILM081, ILM082, ILM083, ILM084, ILM102,ILM103, ILM104, ILM129, ILM133, ILM135, ILM138, ILM142, ILM143, GLX26,GLX40, GLX166, SEX20, SEX76, and SEX180.
 13. A purified proteinpreparation comprising, a Xenorhabdus protein with at least one subunithaving an approximate molecular weight between about 20 kDa to about 350kDa; between about 130 kDa to about 350 kDa; about 80 kDa to about 130kDa; about 40 kDa to about 80 kDa; or about 20 kDa to about 40 kDa. 14.The purified protein preparation of claim 13 comprising, a nativeXenorhabdus protein with at least one subunit having a molecular weightof at least 100 kDa or greater.
 15. A purified protein preparationcomprising a protein containing an amino acid sequence selected from thegroup consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4and SEQ ID NO:5.
 16. A method of controlling an insect comprising,delivering to an insect an effective amount of a protein toxin havingfunctional activity against an insect, wherein the protein is producedby a purified bacterial culture of the genus Xenorhabdus and has annative molecular weight of at least 100 kDa.
 17. The method of claim 16,wherein the Xenorhabdus toxin having functional activity against aninsect is produced by a purified culture of Xenorhabdus nematophilus,Xenorhabdus poinarii, Xenorhabdus bovienii, Xenorhabdus beddingii orXenorhabdus species.
 18. The method of claim 17, wherein said purifiedculture of Xenorhabdus selected from the group consisting of S. carp, X.Wi, X. nem, X. NH3, X. riobravis, GL 133B, DEX1, DEX2, DEX3, DEX4, DEX5,DEX6, DEX7, DEX8, ILM037, ILM039, ILM070, ILM078, ILM079, ILM080,ILM081, ILM082, ILM083, ILM084, ILM102, ILM103, ILM104, ILM129, ILM133,ILM135, ILM138, ILM142, ILM143, GLX26, GLX40, GLX166, SEX20, SEX76, andSEX180.
 19. The method of claim 16, wherein the toxin having functionalactivity against an insect is produced by a purified culture ofXenorhabdus strain designated S. carp, X. Wi, X. nem, X. NH3, X.riobravis, GL 133B, DEX1, DEX2, DEX3, DEX4, DEX5, DEX6, DEX7, DEX8,ILM037, ILM039, ILM070, ILM078, ILM079, ILM080, ILM081, ILM082, ILM083,ILM084, ILM102, ILM103, ILM104, ILM129, ILM133, ILM135, ILM138, ILM142,ILM143, GLX26, GLX40, GLX166, SEX20, SEX76, and SEX180.
 20. The methodof claim 16, wherein the toxin having functional activity against aninsect is a mixture of one or more toxins produced from purifiedcultures of Xenorhabdus.
 21. The method of claim 16, wherein the insectis of the order Coleoptera, Lepidoptera, Diptera, or Acarina.
 22. Themethod of claim 21, wherein the insect species from order Coleoptera areselected from the group consisting of Corn Rootworm, Boll Weevil,wireworms, pollen beetles, flea beetles, seed beetles and Coloradopotato beetle.
 23. The method of claim 21, wherein the insect speciesfrom order Lepidoptera are selected from the group consisting of BeetArmyworm, European Corn Borer, Tobacco Hornworm, Tobacco Budworm,cabbage looper, black cutworm, corn earworm, codling moth, clothes moth,Indian mealmoth, leaf rollers, cabbage worm, cotton bollworm, bagworm,Eastern tent caterpillar, sod webworm and fall armyworm.
 24. The methodof claim 21, wherein the insect species from the order Diptera areselected from the group consisting of pea midge, carrot fly, cabbageroot fly, turnip root fly, onion fly, crane fly, house fly, and variousmosquito species.
 25. The method of claim 21, wherein the insect speciesfrom the order Acarina are selected from the group consisting oftwo-spotted spider mites, strawberry spider mites, broad mites, citrusred mite, European red mite, pear rust mite and tomato russet mite. 26.A method of altering the toxin level or toxin composition produced byXenorhabdus strains comprising, modifying media composition.
 27. Themethod of claim 26 wherein said media composition is modified byfermenting said Xenorhabdus in tryptic soy broth.
 28. The method ofclaim 26 wherein said media composition is modified by increasing ionicstrength of said media.